Homozygous mutant mouse model carrying Msh5 p.D486Y point mutation displayed POI phenotype
The homologous residue for human MSH5 c.G1459 (ENST00 000375755: p.D487) in mouse is Msh5 c.G1456 (EN SM UST00000007250: p.D486), which is highly conserved (Fig. 1C). To examine the functional effect of p.D487Y identified in human POI, the mouse model carrying Msh5 p.D486Y point mutation was generated using a CRISPR/Cas9 system (Supplementary Material, Fig. S3A and B). The homozygous mutant mice Msh5D486Y/D486Y were obtained by intercrossing of Msh5+/D486Y mice (Supplementary Material, Fig. S3C). The homozygous mutant mice were viable at birth and no obvious developmental defects were observed in adults (Fig. 2A). However, all the Msh5D486Y/D486Yfemales were infertile. The size of the ovary was dramatically reduced compared to that of control females (Fig. 2B). The results of histological studies showed that the follicles at different developmental stages (black arrows) were observed in control ovaries (Fig. 2C), whereas no developing follicle was noted in Msh5D486Y/D486Y ovaries (Fig. 2D) at 2 month of age. Numerous Ddx4-positive germ cells (black arrows) were observed in control ovaries (Fig. 2E), but no germ cells were noted in Msh5D486Y/D486Y ovaries (Fig. 2F).
Figure 2 Germ cell loss in ovaries from Msh5D486Y/D486Y mice. (A) The homozygous mutant mice were viable at birth and no obvious developmental defects were observed in adults. (B) The size of ovaries from homozygous mutant females was dramatically reduced compared to the control ovaries. The follicles at different developmental stages (arrows) were observed in control ovaries (C), whereas no developing follicle was noted in Msh5D486Y/D486Y ovaries (D). Numerous Ddx4-positive germ cells (arrows) were observed in control ovaries (E), but no germ cells were noted in Msh5D486Y/D486Y ovaries (F). Scale bars: 200 μm.